Tissue and Metabolic Engineering of Biohybrid Artificial Organs

Abstract

In an effort to develop a biohybrid artificial organ, mammalian cells were engineered with several properties to make them adept in secreting therapeutic proteins and surviving low oxygen levels in an encapsulated environment. Three cell lines, C2C12, Jurkat, and HEK293, were investigated for their ability to secrete human interleukin 2 (hIL2), which can serve as an anti-cancer agent. An intracellular fluorescent protein marker was independently expressed using an internal ribosome entry site sequence so that hIL2 remained active and free for secretion. DsRed fluorescent protein markers were used since red light is known to be transmissible through mammalian tissue. Transient transfection of all three cell lines proved that internal red fluorescence measurements were indeed linearly correlated with the concentration of hIL2 secreted. To increase the survivability of encapsulated cultures, cells were engineered with an anti-apoptotic gene, bcl-2Delta, placed under the control of a hypoxia sensitive promoter. This protective system was found to lessen both hypoxia induced necrosis and apoptosis. To complete the biohybrid system, a novel hydrogel (mTG-Gel), utilizing microbial transglutaminase (mTG) to enyzmatically crosslink gelatin, was developed as a biocompatible cellular scaffold for encapsulating the engineered cells. These gels were stable at physiological temperature (37 ^oC) and could be tailored for enzymatic stability. Specifically, HEK293 cells that were metabolically engineered with all the previous characteristics were encapsulated in 4% mTG-Gels. In situ analysis of DsRed fluorescence showed that cells overlayed with mTG-Gel exhibited reductions in fluorescence with increasing height of gel. Human IL2 diffusion through the hydrogel into overlaying media was found to exhibit the expected dependence on the square of the gel thickness. Diffusion cells were used to determine an effective diffusion coefficient for hIL2, which compared well to that obtained from the gel-overlay cell culture experiments.